>In article <20000221045925.64332.qmail at hotmail.com>, mimiliao at hotmail.com>("mimi Liao") wrote:
>>> Dear All...
>>>> The bacterial plasmids i transfected into mammalian cells are able to
>> replicate in mammalian cells. However i have trouble distinguishing between
>> the bacterial plasmids and the plasmids that have been replicated in the
>> mammalian cells. Does anyone know a good protocol for replication assay ?
>>>> Thanks in advance
>>>> Please reply to mimiliao at hotmail.com>>>> mimi
>>You might wish to try Dpn I digestion to distinguish input plasmid from
>those which have replicated within target cells; input plasmid will be
>digested while replicated should remain untouched.
>>Sorry I don't have a reference handy but a quick scan of medline or pubmed
>should yield something.
This is probably because DpnI cut only methylated site of GATC. Plasmids
grown in dam+ strain E.coli. should be methylated, and hence digested by
DpnI, while replicated plasmids in mammalian cells are not methylated, and
resistant to the digestion.
ZhongLin Chai, PhD
__________________________________________________________
Department of Pathology and Immunology
Monash University Medical School
Alfred Hospital
Commercial Rd, Prahran, VIC 3181, AUSTRALIA
Telephone: (61 3) 9903 0698 (lab)
(61 3) 9903 0696 (office)
Mobile: 0413 58 1940 or International: +61 413 58 1940
Fax: (61 3) 9903 0731
email: zhonglin.chai at med.monash.edu.au
___________________________________________________________
---